Method for producing high potency factor VIII

ABSTRACT

Factor VIII preparations of enhanced potency are obtained from human blood donors by recovering blood from donors who have been treated with vasopressin analogs, such as 1-deamino-6-carba-8-D-arginine vasopressin, in an amount sufficient to increase the circulating blood level of Factor VIII in the donor.

The present invention is concerned with a more concentrated, and hencemore active form of Factor VIII, antihemophilic factor, and with amethod for producing it.

BACKGROUND

It is known that the clotting of human blood is a complicated process,involving a series of reactions mediated by 13 different factors. Italso is well known that a cause of hemophilia is the inability of theafflicted individual to synthesize one of these factors, known variouslyas antihemophilic factor, AHF, AHG or Factor VIII, in amounts sufficientto support adequate clotting. About 40 percent of hemophiliacs have noability to synthesize Factor VIII, while the others have diminishedability. Dried preparations of Factor VIII concentrate are soldcommercially for administration to hemophiliacs for treatment ofbleeding or in advance of surgery. The Factor VIII concentrate isobtained from plasma obtained from human donors, through the use ofknown techniques. At the time of use, the dried concentrate is dissolvedin sterile water, and the resulting solution is administeredintravenously.

The Factor VIII preparation is not pure Factor VIII. Rather, it is aFactor VIII-enriched fraction obtained from plasma and contains othercomponents. It is highly desirable that the Factor VIII concentrate beas pure as possible, but further improvements in purity throughmodification of the procedure for isolating Factor VIII from plasma arenot practically feasible due to the difficulty of separating plasmacomponents.

DESCRIPTION OF THE INVENTION

In accordance with this invention, Factor VIII having increased purityand potency is obtained from the plasma of donors having elevated levelsof Factor VIII obtained by administration to such donor a polypeptideanalog of vasopressin represented by the general formula: ##STR1##wherein W is hydrogen, hydroxy or amino; Y is a residue of analpha-amino acid having a basic aliphatic side chain in theD-configuration containing from 2 to 5 carbon atoms and having on theterminal carbon a basic group such as the amino or guanidino group, suchas arginine, lysine and ornithine; and Z is a disulfide (--S--S--) ormonocarba (--S--CH₂ --) radical; with the proviso that when W ishydroxy, and Y is D-arginine, then Z is monocarba. By the term "residue"of an amino acid is meant the divalent radical formed by removal of thehydrogen atom from the alpha-amino group and the removal of the hydroxylgroup from the carboxyl group of the amino acid. According to acceptedconventions, Phe represents the residue of phenylalanine, Gln representsthe residue of glutamine, Asn represents the residue of asparagine, Prorepresents the residue of proline and Gly represents the residue ofglycine.

Polypeptides within the scope of the general formula (I) are disclosedin U.S. Pat. No. 3,980,631. These, as well as still other polypeptideswithin the scope of the formula, may be synthesized from individualamino acids or amino acid precursors by procedures described in U.S.Pat. No. 3,980,631. The currently preferred polypeptide within theabove-defined class of vasopressin analogs is1-deamino-6-carba-8-D-arginine vasopressin, also known as 6-monocarbadesmopressin or dCDAVP. The preparation of this polypeptide is describedin Example 2 of U.S. Pat. No. 3,980,631.

These polypeptides are known to be pharmaceutically useful asantidiuretics. More recently, however, it has been reported by Mannucciet al in "1-Deamino-8-D-Arginine Vasopressin: A New PharmacologicalApproach to the Management of Haemophilia and Von Willebrand's Disease,"The Lancet, (i) 869 (1977), that when a related vasopressin analog,dDAVP, is administered to those hemophiliacs having diminished abilityto synthesize Factor VIII, the ability to synthesize Factor VIII can beincreased to normal levels.

It has now been discovered in accordance with this invention that theadministration of polypeptide vasopressin analogs defined above tonon-hemophiliacs causes them to synthesize amounts of Factor VIIIsubstantially in excess (e.g., of the order of from about 3 to about 10times) of the normal amount. That is, when the polypeptide isadministered to a normal human, the concentration of Factor VIII in theplasma increases above normal levels by from about 3 to about 10 times.On the other hand, the increased levels of Factor VIII do not cause anundesirable increase in the rate of clotting in normal humans.Apparently this is due, at least in part, to the fact that the levels ofone or more of the other factors involved in clotting are such that theoverall clotting reaction cannot proceed at a rate greater than normal,despite the presence of elevated levels of Factor VIII. In addition, itappears that administration of the polypeptide to humans also increasesthe level of plasminogen activator, which is known to inhibit or reduceclotting. The increased levels of plasminogen activator may serve toprotect against increased clotting. Regardless of theory, however, ithas been shown that the polypeptide can be administered to normal humansat levels which cause substantial increases in the circulating level ofFactor VIII without significant change in clotting and withoutsignificant side-effect other than antidiuresis. This latter effectposes no problem, however, since it is accompanied by inhibition ofthirst and there is no danger of voluntary overhydration.

The amount of polypeptide which is effective to increase the circulatinglevel of Factor VIII ordinarily is an amount sufficient to establish acirculating dosage of polypeptide of about 10 to about 50 μg, andpreferably from about 10 to about 20 μg, in the subject. Thiscorresponds to about 0.2 μg to about 1 μg, and preferably from about 0.2to about 0.4 μg, of polypeptide per kilogram of body weight. This may beachieved directly by intravenous injection, or it may be achievedindirectly by intranasal administration, i.e., in the form of nosedrops. In the latter case, however, the dosage of polypeptide perindividual should be about ten times the desired circulating dosage.Thus, an intranasal dosage of polypeptide from about 100 to about 500μg, and preferably from about 100 to about 200 μg (corresponding to fromabout 2 to about 10, and preferably from about 2 to about 4 μg/kg) isemployed.

The polypeptide is administered in solution in a suitable solvent,preferably water. The solution may contain various additives generallyknown to the art. A preferred medium is physiological saline solution.The solution is preferably acidic having a pH of from about 3 to about5, and especially about 4, to stabilize the polypeptide. It is alsodesirable to include small amounts of bacteriostat, e.g., chlorobutanol,to minimize bacterial contamination in the intranasal preparation.

The concentration of polypeptide in the solution is not narrowlycritical, and can range from about 1 μg/ml to 1000 μg/ml or higher,depending upon the intended mode of administration and dosage. Ingeneral, solutions intended for intranasal applications will containhigher concentrations of polypeptide than solutions intended to beadministered by injection. Thus, solutions for intranasal administrationordinarily will contain from about 100 to about 400 μg polypeptide permilliliter, whereas injectable solutions will contain of the order ofabout 4 to about 10 μg polypeptide per milliliter.

The increase in Factor VIII levels begins within about 15 minutes afteradministration of polypeptide, and increased levels persist for at least4 hours. When the circulating dosage of polypeptide is at least 10 μg,the circulating level of Factor VIII is at least 3 times normal, and maybe 10 or more times normal. Thus, polypeptide is administered to thedonor at least 15 minutes prior to collection, and preferably no morethan 2 hours prior to collection.

The blood of the donor is collected in any conventional manner, and istreated by conventional techniques, for example by freeze-drying, toform a dried Factor VIII preparation. Because of the increasedconcentration of Factor VIII in the blood, the amount of Factor VIII inthe dried preparation recovered from a constant volume of blood iscorrespondingly increased. Furthermore, since the amount of bloodconstituents other than Factor VIII (and plasminogen activator) is notaffected, the proportion of "impurities" in the Factor VIII is reduced.

The following example is illustrative.

Example 1

Intravenous infusions of 10 μg of 1-deamino-6-carba-8-D-argininevasopressin (dCDAVP) were given to each of five consenting healthy malesvolunteers aged 26-40 years. After reclining quietly for 30 minutes,each volunteer was infused through a 15-gauge needle introduced into avein in one antecubital fossa, and blood samples were taken through a15-gauge needle introduced into a vein in the other antecubital fossa. AHarvard constant-infusion pump first administered 25 ml of sodiumchloride solution (154 mmol/1 saline) for 15 minutes, followed by thedCDAVP in 50 ml saline for a further 15 minutes. Blood was sampledbefore, during and after infusion. Patency of the sampling needle wasmaintained by an infusion of saline at 1 ml/min. Pulse rate was recordedthroughout.

Plasma samples which were recovered from the blood samples wereimmediately frozen and stored at -40° C. All samples from a singleinfusion were then thawed and assayed together, usually on the same dayas the infusion, but never more than seven days later. Factor VIIIprocoagulant activity (VIII-AHF) was assayed by a modification of theactiviated partial thromboplastin time using severe hemophilic plasma asthe substrate (Hardisty & MacPherson, Thrombosis et DiathesisHaemorrhagica, 7, 215 (1962), as modified by Veltkamp, M.D. Thesis,University of Leiden, The Netherlands, "Detection of Carrier States inHereditary Coagulation Disorders," Publ. Schatteur-Verlag, Stuttgart(1967)). The assays were standarized against freeze-dried plasma ofknown Factor VIII content, either the 4th or 6th British standard(obtained from the National Institute for Biological Standards andControl, Holly Hill, Hampstead, London). Some samples were also assayedby the two-stage technique of Biggs et al, British Journal ofHaemotology, 1, 20 (1955) using the same Factor VIII standards.

Factor VIII-related antigen (VIII-AGN was assayed by the Laurell method,Scandinavian Journal of Clinical and Laboratory Investigation, 29,Supplement 124, 21 (1972)) using 0.4% antiserum (Behringwerke A.G.,Marburg, Germany in 1% agarose, Indubiose, L-Industrie Biologique,Brancaise, Gennevilliers, France). The buffer used was tris-EDTA-boratepH 8.6 (Aronsonn & Gronwall, Scandinavian Journal of Clinical andLaboratory Investigation, 9, 338 (1957)), diluted five-fold. Resultswere expressed as a percentage of the antigen level in the samefreeze-dried plasma as was used to standardize the bioassay. Crossedimmunoelectrophoresis was carried out by a similar modification of theoriginal technique of Laurell.

The mean Factor VIII-AHF (reported as international units) and the meanFactor VIII-AGN (reported as the percent of standard plasma) reponsesare summarized in tabular form below, together with data obtained insimilar fashion except that saline was substituted for the dCDAVP as acontrol.

    ______________________________________                                               Factor VIII-AHF.sup.(1)                                                                     Factor VIII-AGN.sup.(1)                                  Time, min.                                                                             Saline    dCDAVP    Saline  dCDAVP                                   ______________________________________                                         0       0.98 ± 0.07                                                                          0.99 + 0.11                                                                             117 + 19                                                                              126 + 14                                   71/2   1.03 ± 0.07                                                                          0.97 ± 0.15                                                                          119 ± 19                                                                           129 ± 15                               15.sup.(2)                                                                            0.92 ± 0.08                                                                          1.03 ± 0.08                                                                          128 ± 20                                                                           135 ± 21                              30       0.95 ± 0.06                                                                          1.65 ± 0.11                                                                          125 ± 15                                                                           150 ± 19                              45       0.99 ± 0.09                                                                          2.08 ± 0.16                                                                          128 ± 17                                                                           215 ± 29                              60       0.97 ± 0.09                                                                          1.99 ± 0.16                                                                          131 ± 18                                                                           214 ± 20                              ______________________________________                                         .sup.(1) Values are presented as the mean ± the standard error of the      mean.                                                                         .sup.(2) Onset of infusion.                                              

As is evident, the administration of dCDAVP to the volunteers resultedin a substantial increase in Factor VIII blood levels.

What is claimed is:
 1. In a method for producing a Factor VIIIpreparation comprising collecting blood from a donor, separating theplasma therefrom, and recovering a Factor VIII-rich fraction from saidplasma, the improvement of administering to said donor, in an amounteffective to increase the circulating level of Factor VIII in the bloodof said donor, a polypeptide represented by the formula: ##STR2##wherein W is hydrogen, hydroxy or amino; Y is a residue of analpha-amino acid having a basic aliphatic side chain in theD-configuration containing from 2 to 5 carbon atoms and having on theterminal carbon a basic group; and Z is disulfide or monocarba, with theproviso that when W is hydroxy, and Y is the D-arginine residue, then Zis monocarba, and thereafter recovering blood containing said increasedlevels of Factor VIII.
 2. A method according to claim 1 wherein saidamount is a circulating drug dosage of at least about 10 micrograms. 3.A method according to claim 2 wherein W is hydroxy, Y is the D-arginineresidue and Z is monocarba.
 4. A method according to claim 3 whereinsaid amount is a circulating drug dosage of from about 10 to about 20micrograms.
 5. A method according to claim 2 wherein said administrationis intravenously.
 6. A method according to claim 2 wherein saidadministration is intranasally.